Xiao Wen Tang

Polyhydroxylated C60, fullerenols, as glutamate receptor antagonists and neuroprotective agents

Derivatives of C60 have been shown to be effective free radical scavengers. Hence, many of the biological functions of fullerene are believed to be due to their antioxidant properties. Here we present evidence to show that fullerenols, that are caged fullerene oxides, exert their neuroprotective functions by blocking glutamate receptors and lowering the intracellular calcium, [Ca2+]i. In neuronal cultures, fullerenols reduce glutamate‐induced neurotoxicity by about 80% at 50μM. No significant effect was observed on H2O2/Fe2+‐induced neurotoxicity under the same conditions. Fullerenols were found to inhibit glutamate receptor binding in a dose‐dependent manner inhibiting 50% of glutamate binding at 50 μM. Furthermore, AMPA receptors were found to be more sensitive to fullerenols than NMDA and KA receptors. On the other hand, GABAA receptors and taurine receptors were not significantly affected by fullerenols at the same concentrations used, suggesting that fullerenols inhibit primarily the glutamate receptors. In addition, fullerenols were also found to lower glutamate (Glu) receptor‐induced elevation of [Ca2+]i, suggesting that the underlying mechanism of neuronal protective function of fullerenols is likely due to its ability to block the glutamate receptors and to reduce the level of [Ca2+]i. J. Neurosci. Res. 62:600–607, 2000.

Taurine receptor: kinetic analysis and pharmacological studies.

A new procedure for the preparation of the taurine receptor from mammalian brain is described. The taurine receptor thus prepared shows a Kd of 92 nM, Bmax of 6.0 pmol/mg protein and Hill coefficient of 0.90 suggesting a single site model for the binding of 3H-taurine to the receptor. The binding of 3H-taurine to the receptor is highly specific and is not affected by agonists and antagonists of other receptors such as glutamate, quisqualic acid, kainate and NMDA for the glutamate receptor; glycine and strychnine for the glycine receptor; FNZP for the benzodiazepine receptor; picrotoxin and bicuculline for the GABAB receptor. However, analogues of taurine (e.g., homotaurine and hypotaurine) are potent inhibitors inhibiting more than 50% of 3H-taurine binding at 0.1 microM. Taurine receptor binding is not significantly affected by monovalent cations (e.g., Na+, K+, Li+ and NH4+) at 1 mM or divalent cations (e.g., Mg2+, Ca2+, Ba2+ and Mn2+) at 0.1 mM. However, the binding was completely abolished by Co2+, Zn2+ and Hg2+ at 0.1 mM, suggesting the presence of free sulfhydryl groups near or at the ligand binding site. Among the amino acids tested, cysteic acid was the most potent inhibitor, followed by beta-alanine, valine, tyrosine and cysteine inhibiting 3H-taurine to an extent of 84, 66, 63, 62, and 58% of 1 mM, respectively. Nucleotides and second messengers (e.g., ATP, ADP, cAMP, GTP, cGMP and diacyl glycerol) do not inhibit 3H-taurine binding significantly at 0.1 mM. From above studies, it seems that the taurine receptor is not up- or down-regulated by ions or second messengers at the taurine binding site. Whether the taurine receptor is coupled to a G-protein mediated second messenger system is currently under investigation.

Biphasic Effect of Taurine on Excitatory Amino Acid-Induced Neurotoxicity

In the past, the biological role of taurine as an end product of methionine metabolism and conjugation with bile acids in the liver was considered relatively trivial. However, the physiological role of taurine has received considerable attention since the report by Hayes et al. 7 and Pion et al. 21 that cats fed a taurine-deficient diet developed a central retinal degeneration and a cardiomyopathy. Now, taurine has been proposed to be involved in many important physiological fonctions such as serving as a trophic factor in the development of the CNS (at least in some species)19, 23, 24, maintaining the structural integrity of the membrane17, 20, regulating Ca2+ binding and transport12, 16, and serving as an osmoregulator22, 27, a neuromodulator11 and neurotransmitter14, 15, 18, 25. It is well accepted now that taurine plays an important role in maintaining the integrity of retina and the viability of photoreceptor cells, since absence of dietary taurine in the cat leads to a decrease in taurine concentration in the retina, followed by defects in the electroretinogram, degeneration of the photoreceptors and eventual blindness7, 23.

Regulation of Taurine Biosynthesis and Its Physiological Significance in the Brain

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, was found to be activated under conditions that favor protein phosphorylation and inactivated under conditions favoring protein dephosphorylation. Direct incorporation of 32P into purified CSAD has been demonstrated with [gamma 32P]ATP and PKC, but not PKA. In addition, the 32P labeling of CSAD was inhibited by PKC inhibitors suggesting that PKC is responsible for phosphorylation of CSAD in the brain. Okadaic acid had no effect on CSAD activity at 10 microM suggesting that protein phosphatase-2C (PrP-2C) might be involved in the dephosphorylation of CSAD. Furthermore, it was found that either glutamate- or high K(+)-induced depolarization increased CSAD activity as well as 32P-incorporation into CSAD in neuronal cultures, supporting the notion that the CSAD activity is endogenously regulated by protein phosphorylation in the brain. A model to link neuronal excitation, phosphorylation of CSAD and increase in taurine biosynthesis is proposed.

Mode of Action of Taurine and Regulation Dynamics of Its Synthesis in the CNS

The regulation of taurine biosynthesis can be summarized as following: (i) When neurons are stimulated, the arrival of action potential will open the voltage-dependent Ca2+-channel, resulting in an increase of intracellular free Ca2+, [Ca2+]i, (ii) Elevation of [Ca2+]i will trigger release of taurine as well as activation of PKC, which in turn activates CSAD through protein phosphorylation; (iii) The activated CSAD then synthesizes more taurine to replenish that lost due to stimulation-mediated release; (iv) When intracellular taurine reach a certain level, it then inhibits the activation of PKC directly or indirectly (possibly through regulating Ca2+ availability), thus shutting down activation of CSAD through inhibition of CSAD phosphorylation by PKC; and (v) CSAD soon returns to its inactive state through the action of a protein phosphatase, most likely PrP-2C. The mode of action of taurine inlowering the level of [Ca2+]i, is at least partially due to its inhibition on the reverse mode of Na+-Ca2+ exchanger activity

Isolation of endogenous modulators for the GABAA and taurine receptors

Several endogenous brain substances which inhibit [3H]muscimol binding were isolated, and one of them has been purified to apparent homogeneity. The purification involved the extraction of brain tissue with water, followed by several steps of gel filtration column chromatography and high performance liquid chromatography (HPLC). The muscimol binding inhibitor (MBI) thus obtained appeared to be homogeneous as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at either 220 or 280 nm. Furthermore, the MBI activity coincided with the absorption peak. The purified MBI is not gamma-amino butyric acid (GABA), beta-alanine or taurine since these amino acids are clearly separated from the MBI in the purification procedures. The MBI has no effect on benzodiazepine (BZ) binding or glutamate binding to their respective receptors. However, the MBI is a more potent inhibitor for [3H]taurine binding than that of [3H]muscimol binding. The MBI appears to be a small molecule (< 2000 Da) that is heat and acid/base stable. The chemical nature of the MBI is currently under investigation.

Isolation and characterization of endogenous modulators for GABA system

Pig brain extracts from both soluble and membrane fractions were found to contain potent inhibitors for GABA synthesizing enzyme, GAD, referred to as endogenous GAD inhibitors (EGIs) and for the binding of GABA agonist, muscimol, referred to as muscimol binding inhibitors (MBIs). EGIs and MBIs were first purified through gel-filtration Bio-Gel P-2 columns, in which multiple activity peaks were observed. One of them appears to be co-eluted with eitherl-glutamate or GABA. However, others are clearly separated froml-glutamate or GABA. EGIs were found to be low MW (<1,800 dalton), heat and acid-base stable, negatively charged, non hydrophobic substances. MBIs were found to be low MW (<1,800 dalton) neutral or positively charged substances. MBIs had no effect on [3H]flunitrazepam (FNZP) binding, indicating that they are not endogenous benzodiazepine receptor ligands and they may act specifically on GABA binding site.

Multiplicity of brain cysteine sulfinic acid decarboxylase — Purification, characterization and subunit structure

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in: (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn(2+), methione, and other sulfur-containing amino acids, and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5-20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE, and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 +/- 2 kDa subunits. CSAD appears to require Mn(2+) for its maximum activity. Other divalent cations fail to replace Mn(2+) in activation of CSAD activity. However, the precise role of Mn(2+) in the action of CSAD remains to be determined. Copyright 1996 S. Karger AG, Basel

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABAA receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABAA receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [3H]FNZP binding.

Membrane Associated L-Glutamate Decarboxylase and Insulin-Dependent Diabetes Mellitus (IDDM)

Recently, three novel forms of membrane associated L-glutamate decarboxylase (M-GAD) referred to as M-GADI, M-GADII and M-GADIII were isolated and purified from porcine brain. M-GADI and M-GADII appear to be integral membrane proteins whereas M-GADIII is a peripheral protein attached to membranes in a Ca2+ dependent manner. In addition, M-GADI and M-GADII were found to be the major autoantigens in insulin-dependent diabetes mellitus (IDDM), also known as type 1 diabetes, even more potent than GAD65/GAD67, as demonstrated in immunoprecipitation and immunoblotting tests. M-GADI has a native molecular weight of 96 kDa and consists of two identical subunits of 48 kDa whereas both M-GADII and M-GADIII are homodimers of 60 kDa. M-GADI and M-GADII have fulfilled all three criteria routinely used to determine whether a protein is an integral membrane protein including partitioning in the detergent phase in Triton X-114 partitioning assay, shift in electrophoretic mobility in the presence of ionic detergent and elution by low ionic strength buffer in hydrophobic interaction chromatography. Based on these observations, it is hypothesized that M-GADI and M-GADII may be involved in the pathogenesis of IDDM.